Rapid synthesis of sulfone derivatives as potential anti-infectious agents

Article abstract of DOI:10.1016/j.ejmech.2006.12.015

An original one-pot microwave reaction was developed for the synthesis of sulfone derivatives as new potent antimicrobial agents. This eco-friendly methodology conducted in 30 min led to desired products with good yields. The sulfones (4a and 4b) were obtained via the reaction of 3a with the corresponding halo-derivatives in the presence of sodium hydride. All compounds were tested for their antibacterial and antifungal activities against four bacterial strains (two gram positive, and two gram negative ones) and two yeasts. The disk diffusion method has shown an interesting antibacterial activity for seven compounds (3b-g and 4b) against Staphylococcus aureus. Among these seven compounds, five derivatives (3b-e and 3g) showed activity against Candida tropicalis.

Full text of DOI:10.1016/j.ejmech.2006.12.015

European Journal of Medicinal Chemistry 42 (2007) 880e884
http://www.elsevier.com/locate/ejmech
Short communication
Rapid synthesis of sulfone derivatives as potential anti-infectious agents
C. Curti a, M. Laget b, A. Ortiz Carle b, A. Gellis a, P. Vanelle a,
*
a Laboratoire de Chimie Organique Pharmaceutique, Universite´ de la Me´diterrane´e, UMR CNRS 6517, Faculte´ de Pharmacie, 27 boulevard Jean Moulin,
13385 Marseille Cedex 05, France
b Laboratoire de Parasitologie et Hygie`ne EA 864, Faculte´ de Pharmacie, 27 boulevard Jean Moulin, 13385 Marseille Cedex 05, France
Received 5 September 2006; received in revised form 8 December 2006; accepted 14 December 2006
Available online 9 January 2007
ˆ
Dedicated to the memory of our late Benoıt Celaries
´
`
Abstract
An original one-pot microwave reaction was developed for the synthesis of sulfone derivatives as new potent antimicrobial agents. This eco-
friendly methodology conducted in 30 min led to desired products with good yields. The sulfones (4a and 4b) were obtained via the reaction of
3a with the corresponding halo-derivatives in the presence of sodium hydride.
All compounds were tested for their antibacterial and antifungal activities against four bacterial strains (two gram positive, and two gram
negative ones) and two yeasts. The disk diffusion method has shown an interesting antibacterial activity for seven compounds (3beg and
4b) against Staphylococcus aureus. Among these seven compounds, five derivatives (3bee and 3g) showed activity against Candida tropicalis.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Microwave; Sulfones; Anti-infectious
1. Introduction
sodium sulfite and sodium bicarbonate [7], or sodium sulfite
and disodium hydrogen phosphate [8,9] are often used in the
synthesis of sodium sulfinate derivatives. These sulfinates
could then be easily S-alkylated. Heavy metals in various sol-
vents: Sm/HgCl2 in THF [10], Zn in water [11], In in water
[12], activated Ni [13] were used to catalyze this alkylation
reaction.
Synthesized in 1908 by Fromm and Wittmann [1], dapsone,
bis(4-aminophenyl)sulfone, is still the only representative
member of its pharmacological class (except for acedapsone,
an acetylated prodrug). Dapsone is a well-known anti-infec-
tious agent with antimicrobial [2] and antiparasitic [3,4] activ-
ities and, as sulfonamides, inhibits the synthesis of folic acid
due to structural similarity with p-aminobenzoic acid. It is
also recognized as being effective against several of non-infec-
tious and inflammatory diseases [5]. There are some pieces of
evidence that anti-inflammatory action is not linked to the an-
tibacterial action. In our knowledge, its pharmacomodulation
has never been investigated.
Thus, we report herein the synthesis of new sulfones and
the evaluation of their antibacterial and antifungal activities.
2. Chemistry
Following the extensive development of the use of micro-
wave irradiation in organic synthesis [14e22], a one-pot mi-
crowave assisted synthesis of new aryl-phenacyl sulfones
(3aeh) is hereby presented. Among the different anti-infec-
tious pharmacophores, nitroimidazoles’ and nitrofuranes’ ac-
tivity is clearly dependent on the presence of the nitro
group. In addition, it has been reported that replacement of
the amino group in p-aminobenzoic acid analogues by a nitro
The synthesis of sulfones from sulfonyl chlorides has found
an extensive use in organic synthesis [6]. Mixed salts, such as
* Corresponding author.
E-mail address: patrice.vanelle@pharmacie.univ-mrs.fr (P. Vanelle).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.12.015

C. Curti et al. / European Journal of Medicinal Chemistry 42 (2007) 880e884
881
O
S
O
O
S
O
group does not affect bacteriostatic effect [23] contrary to
most of other substitutions of the amino group. So, we de-
signed to incorporate a nitro group in the sulfone structure,
hoping to improve their biological activity.
water, 100 °C
Na2SO3
NaHCO3
R
R
Cl
R
Na
500 W, 20 min.
1a-g
2a-g
O2N
Thus, a water solution of sodium sulfite, sodium bicarbon-
ate and sulfonyl chlorides (1aeg) was irradiated in a micro-
wave oven at 500 W for 20 min to give the corresponding
sodium sulfinates (2aeg). An ethanolic solution of 2-bromo-
40-nitroacetophenone was then added and the resulting reac-
tion mixture was stirred under irradiation for 10 min to give
the corresponding sulfones (3aeg). Both reaction times and
yields are depicted in Table 1. Also, for comparison purposes,
we have done a parallel synthetic study of sulfones (3aeg)
using a reported method by classical heating conditions
(Table 1). Thus, an aqueous solution of sodium sulfite, sodium
bicarbonate and sulfonyl chloride was heated with stirring at
70e80 ꢀC for 2 h [7]. Then, an ethanolic solution of 2-bromo-
40-nitroacetophenone was added and stirred under reflux was
continued for further 5e6 h to give 3aeg (Scheme 1).
This study revealed that 30 min were needed for the com-
pletion of the reaction under microwave conditions, while
the classical heating conditions required a reaction time be-
tween 5 and 6 h. However, comparable yields were reported
with both operating conditions (Table 1).
Due to the presence of an active methylene group in the
structure of the sulfones (3aeg), these derivatives may be
used as useful intermediates for the synthesis of more complex
structures. For example, when 3a was treated with NaH fol-
lowed by reaction with alkyl halides, such as chloromethyl-
benzene and 2-chloromethylimidazo[1,2-a]pyrazine, the
products obtained were, respectively, 4a and 4b (Scheme 2).
Other reactions leading to different heterocyclic compounds
are in progress.
O
S
O
O
water, 100 °C
O
O2N
Na
500 W, 10 min.
Br
O
S
R
3a-g
O
2a-g
Scheme 1.
and 3g) were active against Candida tropicalis, 3b being the
most effective.
The results of the antimicrobial activity by the disk diffu-
sion method showed an antibacterial activity for several prod-
ucts. Obviously, the antibacterial activity of the sulfones tested
is more pronounced on gram positive than on gram negative
bacteria. The most susceptible strain of bacteria was S. aureus,
and the most susceptible yeast was C. tropicalis. No significant
activity was noted against gram negative bacteria.
The diameters of growth inhibition area for the studied
compounds were in the range 10e28 mm for S. aureus and
12e22 mm for C. tropicalis. Compound 2 showed no activity
against any of the tested strains.
According to the results obtained by the disk diffusion
method, the minimal inhibitory concentration (MIC) values
of the active compounds were determined for the most sensi-
tive organisms. The MIC values obtained confirm the exis-
tence of a significant activity against S. aureus and C.
tropicalis. Among tested compounds, the most potent sulfones
are the dinitro derivative (3c) and the chloronitro derivative
(3e). They showed higher potency than dapsone.
Microplate assay is limited by the water solubility of the
compounds. Thus, it became difficult to determine the absor-
bance of solutions with concentration higher than 0.1 mg/
mL. However, at 0.1 mg/mL concentration in disk diffusion
method, growth inhibition was noted for six derivatives out
of the seven sulfones tested.
Two different alkyl halides were used. This simple and ef-
fective reaction proves the interest of sulfone derivatives as re-
agents for multi-step synthesis.
3. Antimicrobial activity
All the synthesized sulfones were evaluated for their anti-
microbial activity against various strains of bacteria and fungi
using disk diffusion method (Table 2) and microplate assay
method (Table 3).
4. Conclusion
Except compound 4a, all the other sulfones showed good
activity against Staphylococcus aureus (Table 2). The best ac-
tivity was obtained with compound 3c. Five sulfones (3bee
In conclusion, microwave irradiation protocol allowed us to
synthesize sulfones in aqueous solutions with good yields and
in short time. These sulfones (3aeg) were tested for their
Table 1
Classical heating method versus one-pot microwave assisted synthesis of new aryl-phenacyl sulfones
Compound
R
Time (classical heating) hours
Yield (classical heating) %
Time (microwave) min
Yield (microwave) %
3a
3b
3c
3d
3e
3f
C6H5e
5
92
73
63
45
53
64
50
30
30
30
30
30
30
30
90
69
55
42
51
60
44
p-CH3eC6H4e
p-NO2eC6H4e
p-FeC6H4e
p-CleC6H4e
p-IeC6H4e
1-Naphthyl-
5
5.5
6
5.5
6
3g
6

882
C. Curti et al. / European Journal of Medicinal Chemistry 42 (2007) 880e884
Table 3
Minimal inhibitory concentrations using the microdilution method
O2N
O2N
O
O
NaH, N2
DMSO
Compound
MIC (mg/mL)
R'
O
O
R'
Cl
S
O
S
Staphylococcus aureus
Candida tropicalis
O
3b
3c
3d
3e
3f
>0.1
0.1
>0.1
0.1
4
3a
>0.1
0.1
>0.1
0.1
63%
55%
a : R' =
b : R' =
>0.1
>0.1
>0.1
>0.1
>0.1
>0.1
N
N
3g
4b
N
Scheme 2.
Dapsone
Streptomycin
Amphotericin
>0.1
0.025
e
>0.1
e
antimicrobial activity and the results showed that two deriva-
tives (3c and 3e) possess a quantifiable activity.
A QSAR study, which may help us to find the most efficient
structure, in order to improve activity or solubility is currently
in progress.
0.012
5.1.1. General procedure for the microwave assisted
synthesis of sulfone derivatives 3aeg
To a solution of sulfonyl chloride (6 mmol) in water
(15 mL), sodium sulfite (1.26 g, 10 mmol) and sodium bicar-
bonate (0.840 g, 10 mmol) were added. The reaction mixture
was heated under reflux in a microwave oven under irradiation
at 500 W during 20 min. Then, an ethanolic solution of 2-
bromo-40-nitroacetophenone (0.500 g, 2.05 mmol) was added.
Heating of the reaction mixture was continued for 10 min un-
der the same conditions. After cooling, the reaction mixture
was neutralized by adding diluted chlorhydric acid and the
solid product obtained was filtered and crystallized from the
appropriate solvent.
5. Experimental protocols
5.1. Chemistry
Microwave assisted reactions were done in a multimode
microwave oven ETHOS Synth Lab Station (Ethos start, Mile-
stone Inc.). Melting points were determined with a B-540
Buchi melting point apparatus. H NMR (200 MHz) and 13C
1
¨
NMR (50 MHz) spectra were recorded on a Bruker ARX
¨
200 spectrometer in CDCl3 at the service interuniversitaire
1
´
de RMN de la Faculte de Pharmacie de Marseille. H and
13C NMR chemical shifts (d) are reported in parts per million
with respect to CDCl3 7.26 ppm (1H) and 77 ppm (13C). Ele-
mental analysis was carried out at the Centre de Microanalyses
5.1.1.1. 2-Benzenesulfonyl-1-(4-nitrophenyl)-ethanone 3a. Color-
1
less crystals, m.p. 138 ꢀC (ethanol) (Lit.: 136e138 ꢀC [24]), H
´
´ ˆ
de la Faculte des Sciences et Techniques de Saint-Jerome. The
following adsorbent was used for flash column chromatogra-
phy: silica gel 60 (Merck, particle size 0.063e0.200 mm,
70e230 mesh ASTM). TLC was performed on 5 cm ꢁ 10 cm
aluminium plates coated with silica gel 60F254 (Merck) in
appropriate solvent. Analysis indicated by the symbols of
the elements was within ꢂ0.4% of the theoretical values.
NMR (CDCl3), d: 8.34 (d, J ¼ 8.9 Hz, 2H), 8.15 (d, J ¼
8.9 Hz, 2H), 7.88 (d, J ¼ 7.2 Hz, 2H), 7.64 (m, 3H), 4.77 (s,
2H). 13C NMR (CDCl3), d: 64.0 (CH2), 124.0 (2CH), 128.5
(2CH), 129.4 (2CH), 130.5 (2CH), 134.6 (CH), 138.4 (C),
140.0 (C), 186.8 (C]O), CeNO2 not observed in these condi-
tions. Anal. Calcd for C14H11NO5S (305.31): C, 55.08; H,
3.63; N, 4.59. Found: C, 55.25; H, 3.52; N, 4.22.
Table 2
Antibacterial activity by the disc diffusion method, mean of inhibition diameters (mm)
Compound
E. coli
P. aeruginosa
S. aureus
E. hirae
C. albicans
C. tropicalis
DMSO
3b
e
12
e
e
e
e
e
e
e
e
e
34
e
e
e
e
e
e
e
e
e
e
e
24
e
e
e
e
e
e
e
e
e
e
e
e
e
14
e
e
e
e
e
e
e
e
e
e
e
e
e
e
18
e
11
28
25
24
11
10
e
16
27
e
22
12
19
19
e
3c
3d
3e
3f
3g
12
e
e
4a
4b
Streptomycin
Penicillin
Chloramphenicol
Fluconazole
e
e
e
e
e
21
e ¼ No growth inhibition.

C. Curti et al. / European Journal of Medicinal Chemistry 42 (2007) 880e884
883
5.1.1.2. 1-(4-Nitrophenyl)-2-(toluene-4-sulfonyl)-ethanone 3b.
J ¼ 8.9 Hz, 2H), 8.19 (d, J ¼ 7.3 Hz, 1H), 8.17 (d,
J ¼ 8.2 Hz, 1H), 8.05 (d, J ¼ 8.9 Hz, 2H), 7.99 (m, 1H), 7.73
(m, 2H), 7.56 (t, J ¼ 7.7 Hz, 1H), 4.94 (s, 2H). 13C NMR
(CDCl3), d: 63.4 (CH2), 123.5 (CH), 123.9 (2CH), 124.3
(CH), 127.4 (CH), 128.7 (CH), 129.3 (C), 129.4 (CH), 130.3
(2CH), 131.4 (CH), 133.5 (C), 134.2 (C), 136.2 (CH), 140.1
(C), 186.8 (C]O). CeNO2 not observed in these conditions.
Anal. Calcd for C18H13NO5S (355.37): C, 60.84; H, 3.69; N,
3.94. Found: C, 60.46; H, 3.49; N, 4.10.
Pale yellow crystals, m.p. 147 ꢀC (CH2Cl2/hexane) (Lit.:
1
146e147 ꢀC [24]), H NMR (CDCl3), d: 8.34 (d, J ¼ 9.0 Hz,
2H), 8.15 (d, J ¼ 9.0 Hz, 2H), 7.75 (d, J ¼ 7.9 Hz, 2H), 7.37
(d, J ¼ 7.9 Hz, 2H), 4.74 (s, 2H), 2.47 (s, 3H). 13C NMR
(CDCl3), d: 21.7 (CH3), 64.2 (CH2), 124.0 (2CH), 128.5
(2CH), 130.0 (2CH), 130.5 (2CH), 135.4 (C), 140.0 (C),
145.9 (C), 187.0 (C]O), CeNO2 not observed in these con-
ditions. Anal. Calcd for C15H13NO5S (319.33): C, 56.42; H,
4.10; N, 4.39. Found: C, 56.23; H, 4.06; N, 4.17.
5.1.2. General procedure for the preparation of 4a and b
To a solution of 2-benzenesulfonyl-1-(4-nitrophenyl)-etha-
none (0.500 g, 1.63 mmol) in DMSO (15 mL) under inert at-
mosphere, NaH (0.043 g, 1.8 mmol) was added. The reaction
mixture was stirred for 1.5 h and then alkyl halide
(1.8 mmol) in DMSO (2 mL) was added. After completion
of the reaction (TLC), it was diluted with cold water and the
solid precipitate was filtered and purified by flash column
chromatography (eluent: toluene for 4a, petroleum ether/ace-
tone (6:4) for 4b).
5.1.1.3. 2-(4-Nitrobenzenesulfonyl)-1-(4-nitrophenyl)-ethanone
3c. Yellow crystals, m.p. 188 ꢀC (ethanol) (Lit.: 190e192 ꢀC
1
[24]), H NMR (CDCl3), d: 8.45 (d, J ¼ 9.0 Hz, 2H), 8.38 (d,
J ¼ 9.0 Hz, 2H), 8.14 (m, 4H), 4.84 (s, 2H). 13C NMR
(CDCl3), d: 63.4 (CH2), 124.2 (2CH), 124.5 (2CH), 130.3
(2CH), 130.4 (2CH), 139.5 (C), 143.6 (C), 186.5 (C]O),
CeNO2 not observed in these conditions. Anal. Calcd for
C14H10N2O7S (350.30): C, 48.00; H, 2.88; N, 8.00. Found:
C, 48.21; H, 3.05; N, 8.09.
5.1.1.4. 2-(4-Fluorobenzenesulfonyl)-1-(4-nitrophenyl)-etha-
none 3d. Colorless crystals, m.p. 139e140 ꢀC (CH2Cl2/hex-
ane, 3:7), 1H NMR (CDCl3), d: 8.36 (d, J ¼ 9.0 Hz, 2H), 8.16
(d, J ¼ 9.0 Hz, 2H), 7.90 (dd, J ¼ 1.4, 4.9 Hz, 2H), 7.26 (d,
2H), 4.77 (s, 2H). 13C NMR (CDCl3), d: 63.9 (CH2), 116.8
(d, J ¼ 23.0 Hz, 2CH), 124.0 (2CH), 130.4 (2CH), 131.6 (d,
J ¼ 9.9 Hz, 2CH), 134.3 (d, J ¼ 3.3 Hz, C), 139.8 (C), 151.0
(C), 166.3 (d, J ¼ 258.3 Hz, C), 186.9 (C]O), CeNO2 not
observed in these conditions. Anal. Calcd for C14H10FNO5S
(355.37): C, 52.01; H, 3.12; N, 4.33. Found: C, 52.11; H,
3.19; N, 4.29.
5.1.2.1. 2-Benzenesulfonyl-1-(4-nitrophenyl)-3-phenyl-propan-
1-one 4a. Buff crystals, m.p. 171 ꢀC (diethyl ether), 1H
NMR (CDCl3), d: 8.19 (d, J ¼ 9.0 Hz, 2H), 7.84 (m, 4H),
7.73 (m, 1H), 7.60 (m, 3H), 7.14 (m, 3H), 7.05 (m, 1H),
5.28 (m, 1H), 3.39 (m, 2H). 13C NMR (CDCl3), d: 34.2
(CH2), 72.5 (CH), 123.8 (2CH), 127.4 (CH), 128.8 (2CH),
129.0 (2CH), 129.2 (2CH), 129.6 (2CH), 129.8 (2CH), 134.7
(CH), 135.3 (C), 136.1 (C), 141.6 (CH), 150.5 (C), 191.5
(C]O). Anal. Calcd for C21H17NO5S (395.43): C, 63.79; H,
4.33; N, 3.54. Found: C, 63.93; H, 4.38; N, 3.42.
5.1.2.2. 2-Benzenesulfonyl-3-imidazo[1,2-a]pyrazin-2-yl-1-(4-
5.1.1.5. 2-(4-Chlorobenzenesulfonyl)-1-(4-nitrophenyl)-etha-
none 3e. Yellow crystals, m.p. 150 ꢀC (CH2Cl2/hexane)
(Lit.: 156e157 ꢀC [24]), 1H NMR spectrum (CDCl3), d:
8.36 (d, J ¼ 9.0 Hz, 2H), 8.16 (d, J ¼ 9.0 Hz, 2H), 7.82 (d,
J ¼ 8.9 Hz, 2H), 7.56 (d, J ¼ 8.9 Hz, 2H), 4.77 (s, 2H). 13C
NMR spectrum (CDCl3), d: 63.8 (CH2), 124.1 (2CH), 129.8
(2CH), 130.1 (2CH), 130.5 (2CH), 136.7 (C), 139.8 (C),
141.6 (C), 151.0 (CeNO2), 186.8 (C]O). Anal. Calcd for
C14H10ClNO5S (339.75): C, 49.49; H, 2.97; N, 4.12. Found:
C, 49.80; H, 3.12; N, 3.93.
nitrophenyl)-propan-1-one 4b. Colorless crystals, m.p. 156e
1
157 ꢀC (butan-2-ol), H NMR (CDCl3), d: 8.81 (s, 1H), 8.26
(d, J ¼ 9.0 Hz, 2H), 8.12 (d, J ¼ 9.0 Hz, 2H), 7.94 (dd,
J ¼ 1.3, 4.5 Hz, 1H), 7.82 (m, 3H), 7.65 (d, J ¼ 7.3 Hz, 1H),
7.53 (m, 3H), 5.99 (m, 1H), 3.68 (m, 2H). 13C NMR
(CDCl3), d: 26.9 (CH2), 69.2 (CH), 111.4 (CH), 118.5 (CH),
123.7 (2CH), 129.3 (2CH), 129.5 (2CH), 129.9 (CH), 130.1
(2CH), 134.7 (CH), 136.5 (C), 141.5 (C), 142.8 (CH), 143.4
(C), 191.1 (C]O). CeNO2 not observed in these conditions.
Anal. Calcd for C21H16N4O5S (436.44): C, 57.79; H, 3.70; N,
12.84. Found: C, 57.96; H, 3.73; N, 12.37.
5.1.1.6. 2-(4-Iodobenzenesulfonyl)-1-(4-nitrophenyl)-ethanone
3f. Yellow crystals, m.p. 209e210 ꢀC (cyclohexane) (Lit.:
1
217 ꢀC [24]), H NMR (DMSO-d6), d: 8.31 (d, J ¼ 8.6 Hz,
5.2. Antimicrobial activity
2H), 8.16 (d, J ¼ 8.6 Hz, 2H), 8.02 (d, J ¼ 8.1 Hz, 2H), 7.64
(d, J ¼ 8.1 Hz, 2H), 5.50 (s, 2H). 13C NMR (DMSO-d6), d:
62.7 (CH2), 103.2 (C), 123.9 (2CH), 129.9 (2CH), 130.6
(2CH), 138.3 (2CH), 138.9 (C), 140.2 (C), 150.5 (C), 188.7
(C]O). Anal. Calcd for C14H10INO5S (431.20): C, 39.00;
H, 2.34; N, 3.25. Found: C, 38.89; H, 2.10; N, 3.23.
5.2.1. Microorganisms and media
Six reference strains were used in our study.
Escherichia coli ATCC 10536.
Pseudomonas aeruginosa ATCC 15442.
Staphylococcus aureus ATCC 6538.
Enterococcus hirae ATCC 10541.
Candida albicans ATCC 90029.
Candida tropicalis IP 2031.
5.1.1.7. 2-(Naphthalene-1-sulfonyl)-1-(4-nitrophenyl)-ethanone
1
3g. Buff crystals, m.p. 169e170 ꢀC (CH2Cl2/hexane, 1:9), H
NMR (CDCl3), d: 8.76 (d, J ¼ 8.5 Hz, 1H), 8.26 (d,

884
C. Curti et al. / European Journal of Medicinal Chemistry 42 (2007) 880e884
The bacterial strains were grown on Trypticase soy agar
assayed by absorbance measurement at 623 nm. MIC was de-
fined as the lowest concentration of compound allowing no
visible growth.
(Becton Dickinson) at 37 ꢀC for 24 h and the yeast on Sabour-
aud agar for 48 h. Inocula were prepared in TCE (tryptone
0.1%, NaCl 8%, wt/vol) by adjusting the turbidity at 623 nm
to obtain 1e3 ꢁ 105 CFU/mL. The antibacterial and antifun-
gal effects were tested by the disk diffusion method [25] and
the MIC of the more active products was determined by using
a microplate assay.
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Hinton agar medium (MHA, Becton Dickinson) and a top
layer (2 mL) of 0.2% MHA medium inoculated with each bac-
teria suspension (1e3 ꢁ 105 CFU/mL). After drying, 6 mm di-
´
ameter disks (biodisks Merieux) soaked with 10 mL of
different compounds were placed on the agar. Disks contain-
ing streptomycin (10 mg), penicillin G (10 U/IE), chloram-
phenicol (30 mg) and fluconazole (20 mg) were used as
positive controls and DMSO (10 mL) as negative control. All
tests were performed in duplicate, and the antibacterial activity
was expressed as the mean of inhibition diameters (mm)
produced.
[18] C.R. Strauss, Handbook of Green Chemistry and Technology (2002)
397e415.
[19] P.M.D. Mingos, R.D. Baghurst, Microwave-Enhanced Chemistry (1997)
3e53.
5.2.3. Minimal inhibitory concentration
Antimicrobial activity was determined by using a micro-
plate assay [26]. Broth microdilution method was used to de-
termine the MIC and was performed in sterile 96-well
microplates. Each compound (10 mg/mL in DMSO) was
transferred to each microplate well, in order to obtain a two-
[20] A. Loupy (Ed.), Microwaves in Organic Synthesis, Wiley-VCH, Wein-
heim, 2006 and references herein.
[21] P. Lidstrom, J. Tierney, B. Wathey, J. Westman, Tetrahedron 57 (2001)
9225e9283.
[22] S. Caddick, Tetrahedron 51 (1995) 10403e10432.
[23] O.H. Johnson, D.E. Green, R. Pauli, J. Biol. Chem. 153 (1944) 37e47.
[24] A. Borchardt, A. Kopkowski, W. Walilewski, A. Zakrzewski, Prace Wyd-
zialu Nauk Technicznych seria A 18 (1990) 35e41.
fold serial dilution in 100 mL of MuellereHinton broth. The
¨
inocula (100 mL) containing 6 ꢁ 105 CFU of each bacteria
[25] A.W. Bauer, W.M. Kirby, J.C. Sherris, M. Turck, Am. J. Clin. Pathol. 45
(1966) 493e496.
and yeast were added to each well. The final volume was
200 mL of medium MuellereHinton broth. A number of wells
¨
[26] NCCLS (National Committee for Clinical Laboratory Standards), Perfor-
mance Standards for antimicrobial susceptibility testing e Twelfth inter-
national supplement, M100-S12-Wayne, P.A., 2002.
were reserved for sterility control, inoculum viability and
DMSO effect. After incubation for 24 h or 48 h, growth was